Multiple T cell lineages develop in the thymus through well-defined maturation pathways. The maturation of these T lineage cells requires continuous interaction between T cell precursors and the thymic microenvironment, which consists of various stromal cells including thymic cortical and medullary epithelial cells (cTECs and mTECs, respectively). It has been established that cTECs play critical roles in development of CD4- CD8- double negative thymocytes and positive selection while mTECs mediate negative selection and post-positive selection maturation. However, the mechanisms allowing cTECs and mTECs to deliver signals to different stages and different lineages of developing T cells remain unknown. The different fates of developing T cells are likely determined by their interactions with various subpopulations of TECs. It is well established that cTECs and mTECs display extensive heterogeneity. The highly complex nature of TECs strongly supports the hypothesis that individual subsets of TECs perform distinct functions in thymocyte maturation. However, this hypothesis has not been tested because it has not been possible to separate the function of the various cell subpopulations comprising the highly heterogeneous TEC compartment in vivo. To address this issue, we have developed several mouse genetic models that allow the selective deletion of distinct subpopulations of TECs in vivo and in vitro. We have inserted a fusion protein consisting of simian diphtheria toxin receptor- green fluorescent protein into the loci of individual genes expressed in subsets of cTECs or mTECs. In one such model, we show that injection of diphtheria toxin induces rapid deletion of cTECs and massive reduction in thymocytes, demonstrating the effectiveness of this approach in assessing the role of individual TECs in thymocyte maturation. In this proposal, we will investigate the roles of individual TEC subsets in thymocyte maturation using these genetic models. Data obtained from these experiments will provide valuable information about thymocyte-TEC interactions, and generate a framework for identification of novel molecular mechanisms involved in thymocyte development and selection. Importantly, these genetic models are also valuable tools to study cellular regulation of thymic organogenesis and regeneration.Narrative T lymphocytes undergo maturation in the thymus. This process depends on the microenvironment provided by the thymic structure. Thymic epithelial cells are important constituents of the thymic structure and provide indispensable signals for T lymphocyte maturation. Up to this point, how thymic epithelial cells support T lymphocyte maturation is largely unknown. However, understanding the function of thymic epithelial cells in supporting T cell maturation is essential to our understanding of immunodeficiency and autoimmune diseases as dysfunction of thymic epithelial cells causes these diseases. We propose to use genetic mouse models to dissect the function of different populations of thymic epithelial cells in thymocyte maturation. The results from our studies will help us to understand how autoimmune disease and immunodeficiency develop. In addition, these models can also be used to study thymic regeneration after aging.